Pathogens such as Pneumocystis (P), Toxoplasma gondii (Tg)and Mycobacterium avium (Ma) are major causes of opportunistic infection and mortality in immunocompromised patients, particularly those with AIDS. Pneumocystis organisms represent a large group of species of atypical fungi with universal distribution,each with specificity for a specific mammalian host. Pneumocystis jirovecii (pj) is the causative agent of Pneumocystis pneumonia(PcP), one of the most frequent and severe opportunistic infections in immunocompromised patients. Current treatment for PcP combines sulfamethoxazole with trimethoprim, targeting folate biosynthesis. Up to 50% of AIDS patients do not tolerate this treatment long term. Recent studies also show that mutations accumulate over time in the target enzymes, dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), potentially giving rise to drug resistance. These findings underscore the crucial need to develop more effective treatments. A major goal of this project is to structurally and biochemically characterize pjDHFR and its variants in order to design effective inhibitors that have potential as therapeutic agents for the treatment of PcP. Two specific aims are proposed to test the hypothesis that efficacy of antifolate use in combating infections from opportunistic pathogens is the result of specific enzyme-inhibitor interactions with the target DHFR. Specific aim one focuses on cloning, expression, purification and crystallization of pjDHFR in complex with selected enzyme inhibitors. A baculovirusexpression system has been developed to produce soluble, stable enzyme and initial biochemical assays reveal nanomolar inhibitionagainst pjDHFR by a novel antifolate. Structural characterization of this pjDHFR inhibitor complex is underway. Molecular modelingtools will be used for in silico screening of small molecule libraries to define novel scaffolds for synthesis and testing. Computational methods such as 3D QSAR will be used to predict the efficacy of known antifolates for binding to pjDHFR. These data will be used to guide synthesis of novel inhibitors. Th e focus of the second specific aim is to carry out site-directed mutagenesis studies on DHFR to determine the role of specific residues in modulatingpjDHFR inhibitorpotency and in conferring drug-resistance as observed in AIDS patient isolates. Application of novel proteomic tools and homology modeling techniques will be used to determine residues that are critical to enzyme fold and function. These results will help guide the design of species selective inhibitors. Mutagenesis studies will be carried out to test these possibilitiesin the structure-based correlations to help design novel pjDHFRinhibitors.